Mitophagy-promoting miR-138-5p promoter demethylation inhibits pyroptosis in sepsis-associated acute lung injury.

BACKGROUND: The present study was designed to explore the potential regulatory mechanism between mitophagy and pyroptosis during sepsis-associated acute lung injury (ALI).

METHODS: In vitro or in vivo models of sepsis-associated ALI were established by administering lipopolysaccharide (LPS) or performing caecal ligation and puncture (CLP) surgery. Pyroptosis levels were detected by electron microscopy, immunofluorescence, flow cytometry, western blotting and immunohistochemistry. Dual-luciferase reporter gene assay was applied to verify the targeting relationship between miR-138-5p and NLRP3. Methylation-specific PCR and chromatin immunoprecipitation assays were used to determine methylation of the miR-138-5p promoter. Mitophagy levels were examined by transmission electron microscopy and western blotting.

RESULTS: NLRP3 inflammasome silencing alleviated alveolar macrophage (AM) pyroptosis and septic lung injury. In addition, we confirmed the direct targeting relationship between miR-138-5p and NLRP3. Overexpressed miR-138-5p alleviated AM pyroptosis and the pulmonary inflammatory response. Moreover, the decreased expression of miR-138-5p was confirmed to depend on promoter methylation, while inhibition of miR-138-5p promoter methylation attenuated AM pyroptosis and pulmonary inflammation. Here, we discovered that an increased cytoplasmic mtDNA content in sepsis-induced ALI models induced the methylation of the miR-138-5p promoter, thereby decreasing miR-138-5p expression, which may activate the NLRP3 inflammasome and trigger AM pyroptosis. Mitophagy, a form of selective autophagy that clears damaged mitochondria, reduced cytoplasmic mtDNA levels. Furthermore, enhanced mitophagy might suppress miR-138-5p promoter methylation and relieve the pulmonary inflammatory response, changes that were reversed by treatment with isolated mtDNA.

CONCLUSIONS: In summary, our study indicated that mitophagy induced the demethylation of the miR-138-5p promoter, which may subsequently inhibit NLRP3 inflammasome, AM pyroptosis and inflammation in sepsis-induced lung injury. These findings may provide a promising therapeutic target for sepsis-associated ALI.